MOLECULAR BIOLOGY OF TROPICAL INFECTIOUS DISEASESdr. Tri Wibawa, PhDDepartment of MicrobiologyGadjah Mada Sch. of. Med

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Eukaryote versus prokaryote

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Watson and Cricks model for DNA replication

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Eukaryotes genomes; Divided into two or more linear DNA molecules. Each contained in a different chromosome. Possess smaller, usually circular, mitochondrial genomes. Third genome located in the chloroplast (in plant and other photosynthetic organism) 10 Mb 100,000 Mb in length. Higher eukaryotes need larger genome to accommodate the extra genes. Correlation between genome size and complexity (???) --- C-value paradox. Space is saved in the gnomes of less complex organism because the genes are more closely packed together.

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Prokaryotes genomes: Whole prokaryotes genomes are smaller than eukaryotes. Most if not all -- are contained in a single DNA molecules. The molecule is circular. Have second circular or linear genome, called PLASMID. Have fewer genes More compact genome organization, more gene but less space. There is NO INTRONS (some exception is in archaea.) Infrequency of repetitive sequences.

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5533exonexonintronupstreamdownstreamInitiation codontermination codon

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Circular shape of microbial DNA

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More than 500 viruses could fit inside a single bacterial cell.Eukaryotes, Prokaryotes, and Virus Size

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Virion Structure

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VIRAL SHAPE

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Helical VirusesMeasles VirusRabies Virus

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IcosahedronIcosahedron polyhedron with 20 triangular facespoliovirusHerpes virusparvovirus

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ComplexsmallpoxInfluenza virusBacteriophage

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Components of VirusesGenome nucleic acid core of the virusConsists of either DNA or RNA

Capsid outer protein coat of the virusFormed from smaller protein units called capsomeres

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Components of Viruses3. Envelope Enclosing structure, similar to the membrane that encloses a cellAcquired at last stage of replication

4. Spikes Projections from envelopeHelps virus contact host cell

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VIRUS GENOMES Is more varied than any other kingdoms Single / double strand Linear / circular / segmented

Single strand virus genome may be: Positive (+) sense (the same polarity as mRNA) Negative (-) sense (need virus-specific polymerase) ambisense (mixture of the two)

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(+) and (-) Sense(+) RNA and (+) DNA have the same sequence as the mRNA (except that in DNA thymine replaces uracil). () RNA and () DNA have the sequence complementary to the mRNA (except that in DNA thymine replaces uracil).Most of these viruses have either a (+) or a () strand genome. Some ssDNA viruses and some ssRNA viruses have ambisense genomes.

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Replication of DNA Genomes

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Replication of RNA Genomes

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Reverse-TranscribingViruses

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Locations of virus genome replication in eukaryotic cells

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Locations of virus genome replication in eukaryotic cells

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Infection Transfection Infectious virus particleInfectious virus particleHost cellsInfectiousVirus particlePurified Nucleic acids(DNA or (+)RNA)

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Influenza virusGemini virusSegmented8 segments negative-sense RNABipartiteTwo molecules Ds-DNA

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DNA viruses

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RNA viruses

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Replication of viruses :

Attachment, penetration and un-coating Expression of viral genome Synthesis of viral component Morphogenesis and releaseSemliki forest virus

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Obligate intracellular parasites

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Segmen genome berada di dalam sel host naked & freePeran HA pada proses infeksiProses replikasi Virus Influenza

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BIOLOGY OF THE FUNGI Somatic structures: Mold form Yeast form DimorphicYeast formMold form

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M. tuberculosis

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Molecular basis of antimicrobial resistanceMechanisms of antimicrobial resistance: The presence of inactivation enzyme Alternative protein for the target of antimicrobial agent Mutation in the target Post-transciptional and post-translational modification of the target. Reduction of the antimicrobial uptake. Active efflux of antimicrobial agent. Over-expression of the target. Unknown mechanism

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Rifampisin (RIF) Action: Inhibition of DNA-dependent RNA polymerase. RNA polymerase: 4 subunit: ; ; ; Genes : rpoA; rpoB; rpoC; rpoD Target: bind to the subunit resulting in transcription inhibition Mutation in the rpoB gene responsible to rifampisin resistance.

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Identified mutations in rpoB genes of MTB

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Mutation in more than one residue of the rpoB gene.

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Isoniazid (INH) Action and target: Not clearly known Candidate; INH or INH metabolite block the synthesis of mycolic acids. Genes : katG. Encoding catalase-peroxidase enzym INH resistance MTB had decreased catalase activity. Mutation in the katG gene responsible to isoniazid resistance.

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Isoniazid (INH)

Genes : inhA. Encoding protein for fatty acid biosynthesis. inhA has correlation with resistance to INH and ETH Polymorphisms were found in the upstream of orfI

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Ethambutol (EMB) Action and target: Not clearly known Candidate;

Inhibition of RNA metabolismInhibition of phospholipid synthesisInhibition of transfer of mycolic acidInhibition of spermidine synthesisInhibition of first step of glucose conversion.

Genes : No genes were identified.

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Pyrazinamide (PZA) Action and target: Not clearly known Candidate; Pyrazinamidase convert PZA to pyarzinoic acid. PZA-resistance MTB lack of the pyrazinamidase activity. Genes : No gene were identified

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Molecular Diagnostic Techniques of TuberculosisMolecular Diagnostic Techniques are indicated: Detection of organisms that cannot be grown in vitro or for which current culture techniques are too insensitive. Require complex media and prolong incubation time.Molecular Diagnostic Techniques consideration: high sensitivity high specificity speed simplicity clinical relevance.

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Basic principle of any Molecular Diagnostic Techniques is the detection of specific nucleic acid (DNA) sequence of the pathogens.

Example: Polymerase chain reaction (PCR) Southern blot hybridization Nested PCR Multiplex PCR Reverse transcriptase PCR (RT PCR) Trancription-mediated amplification (TMA) Ligase chain reaction (LCR) Nucleic acid sequence-based amplification (NASBA)

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PCR versus Conventional diagnostic proceduresAt present: PCR cannot replace the conventionalReason: Sensitivity Specificity Specimen still need to be culture for susceptibility test. Cost

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Avian influenza

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Timeline of Emergence of Influenza A Viruses in Humans191819571968197719971998/92003H1H1H3H2H7H5H5H9SpanishInfluenzaH1N1AsianInfluenzaH2N2RussianInfluenzaAvianInfluenzaHong KongInfluenzaH3N2

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Antigenic DriftAntigenic drift is the natural mutation over time of known strains of influenza to evade the immune system. Antigenic drift occurs in all types of influenza including influenza A, B and C. The same subtype of viruses

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MUTASI VIRUS INFLUENZA Virus Influenza tidak memiliki mekanisme: Proofreading Repair of errors yang terjadi saat replikasi Akibat tidak adanya koreksi thd kesalahan : Komposisi genetik dari virus berubah pada saat bereplikasi pada host. Strain yang beredar berganti dengan varian virus baru dengan sifat antigenik yang berbeda. Perubahan yang biasanya kecil ini konstan, permanet dan biasanya kecil ini merubah komposisi antigenik virus influenz A.

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MutationIn general mutations are changes to the DNA or RNA (genetic materials) of viruses. Mutations can be caused by :Copying errors in the genetic material during cell division Exposure to elements such as radiation, chemicals, viruses or can occur deliberately under cellular control during the processes such as meiosis or hyper-mutation. Mutations are considered the driving force of evolutionLess favorable (or deleterious) mutations are removed from the gene pool by natural selection.Favorable (beneficial or advantageous) ones tend to accumulate.

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Mutation (in humans)Source: WHO/WPRO

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Influenza Surface ProteinsNeuraminidaseHemagglutininRNAM2 protein(only on type A)

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Antigenic DriftRNAHemagglutininNeuraminidaseAntibodiesSialic acid

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Antigenic Shift:Antigenic shift is the process by which two different strains of influenza combine to form a new subtype having a mixture of the surface antigens of the two original strains. The term antigenic shift is specific to the influenza literature; in other viral systems, the same process is called reassortment or viral shift.Antigenic shift occurs only in influenza A because it infects more than just humans. Affected species include other mammals and birds, giving influenza A the opportunity for a major reorganization of surface antigens. New subtype of virus developed

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Reassortment

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Reassortment (in humans)Migratory water birds Source: WHO/WPRO

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Reassortment (in pigs)Migratory water birds Source: WHO/WPRODomestic bird

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From birds to humansMigratory water birds Hong Kong, SAR China 1997, H5N1 Hong Kong, SAR China 1999, H9N2 The Netherlands 2003, H7N7 Hong Kong, SAR China 2003, H5N1Source: WHO/WPRO

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Antigenic Shift

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This overhead shows the standard features found in some (but not all) viruses. A virus particle is essentially a piece of nucleic acid surrounded by a protein coat. The protein coat (i.e. the capsid) is a delivery system for transferring the virus genome from one cell to another. The protein serves to: Provide protection to the nucleic acid against the environment - e.g. nucleases etc. Function in receptor recognition - targeting a virus to a susceptible host and cell type. Surrounding this coat there may be a lipid envelope - this envelope is derived from one of the cell membranes and is not determined by the virus. There may be some modification to the lipid composition induced during virus maturation. Inserted into the lipid envelope there are usually virus proteins which are present as spike projections - these are normally glycoproteins.Due to restrictions on the coding size of many virus genomes the capsid of the virion is made up of repeating subunits, which coat the virus genomic nucleic acid. The redundancy also allows for the fact that if there is an inactivation of part of the capsid the virus does not completely lose its infectivity For example the poliovirus RNA (7kb) can specific at most 250,000 Daltons of protein altogether (some must be used for replication) but the poliovirus virion capsid weighs 6 x 106 Daltons.

Genomic Nucleic AcidViruses only possess a single type of genomic nucleic acid either DNA or RNA but not both. This nucleic acid can be in a variety of physicla forms that can be used as a valuable classification feature.Slide 11Lecture NotesSlide 15Lecture NotesSlide 16Lecture Notes